We first analyzed the mitochondrial electron transport chain (ETC) complexes by Blue-Native-PAGE (BN-PAGE) and found no difference in ETC complex assembly between the CM-K4KO and MHC-Cre groups at baseline (Fig. 1A). As protein acetylation has been shown to negatively affect mitochondrial function, we subjected cardiac mitochondrial proteins to Western blot analysis for acetyl-Lysine to assess overall protein acetylation levels in the mitochondria. As shown in Fig. 1B, there was marked increase of protein acetylation in the CM-K4KO group. Pressure overload induced by transverse aortic constriction (TAC) further enhanced mitochondrial protein acetylation and the same hyperacetylation in CM-K4KO group persisted even after TAC (Fig. 1C). Such hyperacetylation of mitochondrial proteins was also associated with TAC-induced heart failure in C57BL6 WT mice (Fig. 1D), sug- gesting a plausible relationship between hyperacetylation of cardiac mitochondrial proteins and cardiac dysfunction.

To gain a further understanding of these hyper-acetylated proteins, we immunoprecipitated all acetylated mitochondrial proteins by the anti-acetyl-Lysine antibody and probed for Superoxide dismutase 2 (SOD2), Cyclophilin D (CypD) and long chain Acyl-CoA dehydrogenase (LCAD), all of which are known Sirt3 targets and can be acetylated under certain conditions [12,21–23]. As shown in Fig. 1E, all three acetylated proteins were enriched in the CM-K4KO group. Previous studies have reported that hyperacetylation of these proteins are asso- ciated with mitochondrial dysfunction and heart failure [12,21–23]. As such, our data suggested that mitochondria in the KLF4-deficient car- diomyocytes were likely predisposed to hyperacetylation-related de- fects. Previously we showed impaired mitochondrial metabolic function in the KLF4-deficient heart [20], which can be partly attributed to protein hyperacetylation.

Collectively, these data demonstrate that cardiac KLF4-deficiency led to hyperacetylation of mitochondrial proteins, likely due to reduced deacetylation function. Such posttranslational modification of mi- tochondrial proteins can profoundly impair mitochondrial metabolic function at baseline, which may predispose the CM-K4KO hearts vul- nerable to stress [18,20].

Fig. 5. Administration of NMN protected cardiac mitochondria from TAC-induced damage.
(A) EM images from PBS (vehicle) treated hearts after 5 days of TAC.
(B) EM images from NMN (500 mg/kg/day) treated hearts after 5 days of TAC. Scale bar: 1 um. Arrows indicate damaged or abnormal mitochondria. Each image was from individual animal but different areas were chosen to display different phenotype. n = 3 in each group.

3.2. Reduction of Sirt3, NAD+ and NAMPT in the KLF4-deficient heart

Mitochondrial protein acetylation is mainly regulated mainly by micotinamide adenine dinucleotide (NAD+) and the Sirtuin family of NAD+-dependent deacetylases. Among all known mammalian Sirtuin genes, we found the mRNA levels of Sirt3 and Sirt5 were reduced in CM-K4KO heart (Fig. 2A). Of note, Sirt3 is the key deacetylase in the mitochondrial matrix while Sirt5 is more of a deacylase rather than a deacetylase [24]. Hence, we focused on Sirt3 and found its protein le- vels were also reduced in the CM-K4KO mitochondria (Fig. 2B). Al- though it is hard to determine if such subtle decrease of Sirt3 protein is rate-limiting, undoubtably Sirt3 insufficiency can contribute to mi- tochondrial protein hyperacetylation [25].

Since NAD+ is critical for protein acetylation as well as mitochon- drial respiration function, we next sought to determine the NAD+ levels in cardiac tissue. NAD+ levels were reduced in CM-K4KO hearts and pressure overload further affect myocardial NAD+ levels (Fig. 2C). Not surprisingly, TAC reduced cardiac NAD+ levels to much lower levels in the CM-K4KO group (Fig. 2C). NAD+ biosynthesis relies on two path- ways, namely de novo and salvage pathways (Fig. 2D). Because adult heart does not express TDO (tryptophan 2,3-dioxygenase) or IDO (In- doleamine 2,3-dioxygenase), two rate-limiting enzymes in the de novo pathway, nearly all cardiac NAD+ is generated from the salvage pathway [26]. Nicotinamide phosphoribosyltransferase (NAMPT), the key enzyme in NAD+ salvage pathway, was significantly lower in the

CM-K4KO heart and it went further down after TAC (Fig. 2E). These data strongly suggested a NAD+ deficient stage in the CM-K4KO heart, especially after the onset of pressure overload.

The combination of reduced expression of Sirt3 and low NAD+ concentration could result in overall deacetylase activity deficiency in the mitochondria leading to mitochondrial protein hyperacetylation. Moreover, NAD+ is a key coenzyme of the tricarboxylic acid (TCA) cycle and fatty acid β-oxidation, a depleted NAD+ pool would also impair mitochondrial metabolism.

Given the mitochondrial hyperacetylation and NAD+ deficiency observed in the CM-K4KO hearts, we asked if NAD+ repletion could be beneficial to these animals. To bypass the NAMPT defect, we chose to use Nicotinamide Mononucleotide (NMN) as the exogenous NAD+ precursor (Fig. 2D). As expected, NMN administration increased cardiac tissue NAD+ level (Fig. 3A). Further, NMN normalized the mitochon- drial protein acetylation levels in the hearts (Fig. 3B). Strikingly, ad- ministration of NMN at 500 mg/kg/day starting from one day before TAC profoundly preserved the cardiac contractile function and com- pletely rescued the heart failure phenotype in the CM-K4KO group (Fig. 3C). NMN treatment did not affect the expression of hypertrophic genes (i.e. ANF, β-MHC) but blunted the induction of inflammatory genes (i.e. IL1β, CCL2 and IL6) in CM-K4KO hearts (Fig. 3D), indicating that administration of NMN maintained a normal hypertrophic re- sponse to pressure overload but reduced cardiac injury. Due to high mortality rate of CM-K4KO mice after TAC [18], the phenotypical comparison between NMN and PBS (Vehicle) administration were performed within 5 days post-TAC but the CM-K4KO animals with T- AC + NMN administration exhibited 100% survival rate during the course of study (data not shown).

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• People who have AIDS may live longer with the help of acyclovir, an antiviral drug better known for treating herpes. In an ongoing study involving gay and bisexual men with AIDS, researchers found that patients taking acyclovir plus AZT live eight to 15 months longer than those taking just AZT. (AZT by itself generally extends life by about 12 months.) “This is the best news we’ve had about survival with AIDS since AZT became available,” says Dr. Neil Graham, a Johns Hopkins epidemiologist involved with the study. “It’s the first evidence that combination therapy offers survival benefits above and beyond giving just one drug.”
• A fourth AIDS drug has received Food and Drug Administration (FDA) approval. Stavudine (D4T), which interferes with HIV replication in the same manner as the three other AIDS drugs (AZT, ddI and ddC), will be given to patients not helped by those drugs. Bristol-Myers Squibb will sell the drug under the brand name Zerit, which will sell for $6.22 per daily dose. (Some patients have already obtained stavudine through an FDA program that makes promising drugs for life-threatening diseases available before approval.)
• Large-scale clinical trials of two preventive AIDS vaccines have been postponed. Though a committee for the National Institute of Allergies and Infectious Diseases tentatively recommended in April that the trials go ahead, it reconsidered after reports that several volunteers in smaller vaccine trials had become infected with HIV. The volunteers apparently got infected from high-risk behavior (the vaccines themselves can’t cause infection because they consist mainly of protein fragments, not infectious material). Even before the smaller trials started, some scientists were concerned that the vaccines would not protect against all strains and would not confer long-lasting immunity.
• Researchers have discovered one of the steps by which HIV infects cells—and perhaps a way to prevent the infection. After the virus hooks onto a receptor on the surface of a white blood cell, an enzyme on the cell helps the virus get in. By blocking the enzyme, researchers at Boston University School of Medicine prevented the virus from entering cells, at least in test tube experiments. “It’s a totally different approach to intercepting the infective process,” says cell biologist Richard Mandel, one of the researchers. Most AIDS drugs, including AZT, interfere with HIV replication inside infected cells.
• Should home testing kits for AIDS be allowed on the market? The FDA has held a hearing to consider the controversial question. With such a kit, a person smears a few drops of blood on special filter paper at home and mails it to a lab. A week later the person calls a toll-free number for the result and counseling, if necessary, without ever setting foot in a clinic or releasing his or her name.
Proponents think many people who wouldn’t go to clinics might be willing to get tested at home. But representatives of some AIDS clinics worry that people who test positive wouldn’t receive adequate counseling. An informal poll of the FDA drug advisory committee found that most members thought the public health benefits outweighed the risks. Kits would probably sell for $30 to $50 each.

For many people struggling to achieve their weight loss goals, diet fads and appetite suppressants with unpleasant side effects can cause a roller coaster effect of constantly loosing weight only to put it back on. This situation can be highly discouraging and can convince many people that they are never going to reach their goals because they just don’t have what it takes, but this is just not true. If this sounds familiar to you then it may be time to stop following the fads and taking harsh unnatural chemicals, and go with a more natural and effective approach.

The Dr Oz stamp of approval

A supplement called 5htp has been steadily gaining in popularity for some time now and has even been recommended by the highly respected Doctor Oz as a safe and effective tool to help you shed unwanted pounds. This supplement is a chemical that your body naturally produces, so you are not introducing a foreign chemical to your body that may cause unpleasant side effects. It has been shown to be effective in clinical trials that showed participants lost considerably more weight using it than the placebo group lost during the same time period. Doctor Oz has featured this highly effective supplement on his show and his website.

Stops overeating, naturally

One of the reasons this supplement is getting such rave reviews is because the way it works takes many of the common reasons people fail to stick to their diets out of the picture. In fact it will actually help you loose weight without feeling like you are on a diet at all because it helps your body naturally produce chemicals that reduce your appetite. That way you don’t have to force yourself to eat less, you just don’t get as hungry and end up eating less naturally. You will simply have a smaller appetite, which can translate into effortless weight loss.

Improves mood and sleep

Not only does 5htp help control your appetite, it can also help improve your mood and help you sleep better. The chemical in this supplement is part of what the body needs to make serotonin, but unlike serotonin supplements, 5htp can actually cross into the brain so it is more effective. It helps increase the amount of serotonin in your body, which provides a host of great benefits including better sleep, reduced appetite, improved mood, and much more. Best of all this is a naturally occurring chemical in your body so you don’t have to worry about it being harmful.

Some minor side effects to watch out for

While it is a naturally occurring chemical in the body, there are a few things you should keep in mind if you want to take this supplement. It may take a couple of weeks before you really start feeling the results, but you should not take it for more than 12 weeks at a time. If you have gastrointestinal issues, are taking an anti-depressant, or are pregnant or nursing you must talk to your doctor before beginning to take this supplement.

Our recommendation

Studies have shown 5htp is safe and effective at controlling binge eating at 50-100mg dosages, twice per day. Just make sure to get from a supplier you trust like AlivebyNature.org